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Questions and Answers
Which of the following enzymes is MOST directly involved in repairing double-strand breaks in DNA through homologous recombination?
Which of the following enzymes is MOST directly involved in repairing double-strand breaks in DNA through homologous recombination?
- RecA recombinase (correct)
- Excinuclease
- DNA photolyase
- DNA glycosylase
During double-strand break repair, what is the initial step facilitated by helicases and nucleases?
During double-strand break repair, what is the initial step facilitated by helicases and nucleases?
- DNA synthesis using the homologous strand
- Strand invasion
- End-processing to create 3' overhangs (correct)
- Formation of a D-loop
What enzymatic activity is associated with the RecBCD complex in E. coli?
What enzymatic activity is associated with the RecBCD complex in E. coli?
- Both helicase and nuclease activities (correct)
- Recombinase activity
- Exonuclease activity only
- Helicase activity only
Which component of the RecBCD complex is responsible for recognizing chi sites?
Which component of the RecBCD complex is responsible for recognizing chi sites?
What is the function of RecA in homologous recombination?
What is the function of RecA in homologous recombination?
In Synthesis-Dependent Strand Annealing (SDSA), what is the role of a helicase?
In Synthesis-Dependent Strand Annealing (SDSA), what is the role of a helicase?
What is required for the formation and resolution of Holliday junctions?
What is required for the formation and resolution of Holliday junctions?
What is the direct role of the RuvAB complex in resolving Holliday junctions?
What is the direct role of the RuvAB complex in resolving Holliday junctions?
What is the function of RuvC in resolving Holliday junctions during homologous recombination in E. coli?
What is the function of RuvC in resolving Holliday junctions during homologous recombination in E. coli?
Which of the following proteins is NOT directly involved in bacterial recombinational DNA repair?
Which of the following proteins is NOT directly involved in bacterial recombinational DNA repair?
Which of the following describes the immediate effect of DNA-bound RecA on LexA during the SOS response in E. coli?
Which of the following describes the immediate effect of DNA-bound RecA on LexA during the SOS response in E. coli?
What process is MOST directly facilitated by double-strand break repair during meiosis I?
What process is MOST directly facilitated by double-strand break repair during meiosis I?
During homologous recombination, what ensures the precise alignment needed for crossing over to avoid frameshift mutations?
During homologous recombination, what ensures the precise alignment needed for crossing over to avoid frameshift mutations?
What is the function of Spo11 in initiating homologous recombination?
What is the function of Spo11 in initiating homologous recombination?
What is the role of Resolvases during meiotic recombination?
What is the role of Resolvases during meiotic recombination?
What is the MOST immediate consequence of gene conversion?
What is the MOST immediate consequence of gene conversion?
What can happen if the replication fork arrives at a point on the DNA before damage is repaired?
What can happen if the replication fork arrives at a point on the DNA before damage is repaired?
What is the primary role of translesion DNA synthesis (TLS)?
What is the primary role of translesion DNA synthesis (TLS)?
Under what circumstances is the replicative DNA polymerase more likely to hop over damage on the lagging strand?
Under what circumstances is the replicative DNA polymerase more likely to hop over damage on the lagging strand?
What is a likely outcome when DNA polymerase encounters a single-strand region?
What is a likely outcome when DNA polymerase encounters a single-strand region?
What is the purpose of fork regression in response to a DNA lesion?
What is the purpose of fork regression in response to a DNA lesion?
What typically happens after fork regression during DNA replication?
What typically happens after fork regression during DNA replication?
Besides homologous recombination, which mechanism can cells use to rejoin the ends of a double-strand break?
Besides homologous recombination, which mechanism can cells use to rejoin the ends of a double-strand break?
What is a key characteristic of nonhomologous end joining (NHEJ)?
What is a key characteristic of nonhomologous end joining (NHEJ)?
How does CRISPR-Cas9 enable precise genome editing?
How does CRISPR-Cas9 enable precise genome editing?
What is required in order to replace one allele with another using CRISPR/Cas9?
What is required in order to replace one allele with another using CRISPR/Cas9?
What effect does DNA-bound RecA have on LexA during the SOS response in E. coli?
What effect does DNA-bound RecA have on LexA during the SOS response in E. coli?
During homologous recombination, what process ensures the precise alignment needed for crossing over to avoid frameshift mutations?
During homologous recombination, what process ensures the precise alignment needed for crossing over to avoid frameshift mutations?
Based on the information, what is RecA's MOST direct interactions?
Based on the information, what is RecA's MOST direct interactions?
A researcher is studying DNA repair mechanisms in E. coli. They observe a strain with a mutation that impairs the cell's ability to repair double-strand breaks. Which of the following enzymes is MOST likely affected by this mutation?
A researcher is studying DNA repair mechanisms in E. coli. They observe a strain with a mutation that impairs the cell's ability to repair double-strand breaks. Which of the following enzymes is MOST likely affected by this mutation?
A scientist is investigating the initial steps of double-strand break repair. They discover a mutation that prevents the formation of 3' overhangs. This mutation MOST likely affects the function of which type of enzyme?
A scientist is investigating the initial steps of double-strand break repair. They discover a mutation that prevents the formation of 3' overhangs. This mutation MOST likely affects the function of which type of enzyme?
A researcher observes that a bacterial strain is deficient in recognizing chi sites during homologous recombination. This deficiency is MOST likely due to a mutation affecting which component of the RecBCD complex?
A researcher observes that a bacterial strain is deficient in recognizing chi sites during homologous recombination. This deficiency is MOST likely due to a mutation affecting which component of the RecBCD complex?
In a mutant strain of bacteria, the RecA protein is unable to bind to single-stranded DNA. Which of the following processes would be MOST directly affected in this strain?
In a mutant strain of bacteria, the RecA protein is unable to bind to single-stranded DNA. Which of the following processes would be MOST directly affected in this strain?
A researcher is studying the Synthesis-Dependent Strand Annealing (SDSA) pathway. They observe that newly synthesized DNA strands are unable to separate from the template strand. This is MOST likely due to a defect in which type of enzyme?
A researcher is studying the Synthesis-Dependent Strand Annealing (SDSA) pathway. They observe that newly synthesized DNA strands are unable to separate from the template strand. This is MOST likely due to a defect in which type of enzyme?
Which of the following steps is essential for the formation of Holliday junctions and is a critical component of homologous recombination?
Which of the following steps is essential for the formation of Holliday junctions and is a critical component of homologous recombination?
If the RuvB component of the RuvAB complex is non-functional, how would this MOST directly affect Holliday junction?
If the RuvB component of the RuvAB complex is non-functional, how would this MOST directly affect Holliday junction?
A research team is investigating the Holliday Junctions during homologous recombination in. Their findings suggest a non-functional Dimer formation, which option would be MOST affected?
A research team is investigating the Holliday Junctions during homologous recombination in. Their findings suggest a non-functional Dimer formation, which option would be MOST affected?
Which of the following is not one of RecBCD's activity?
Which of the following is not one of RecBCD's activity?
During the SOS response in E. coli, DNA-bound RecA results in which effect to LexA:
During the SOS response in E. coli, DNA-bound RecA results in which effect to LexA:
Flashcards
Double-strand break repair
Double-strand break repair
DNA damage repair mechanism for double-strand breaks, using RecA recombinase.
RecBCD
RecBCD
Enzyme that binds to dsDNA ends and moves inward, unwinding DNA and degrading it.
RecA
RecA
A protein that promotes strand invasion during homologous recombination.
SDSA (Synthesis-Dependent Strand Annealing)
SDSA (Synthesis-Dependent Strand Annealing)
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Holliday Junctions
Holliday Junctions
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RuvAB complex
RuvAB complex
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RuvC
RuvC
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RecG
RecG
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Gene Conversion
Gene Conversion
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Translesion Synthesis (TLS)
Translesion Synthesis (TLS)
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CRISPR/Cas9
CRISPR/Cas9
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Nonhomologous End Joining
Nonhomologous End Joining
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Adaptive Evolution
Adaptive Evolution
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SOS response
SOS response
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Study Notes
- DNA double-strand break repair and homologous recombination are essential for genome maintenance
Double-Strand Break Repair
- Double-strand breaks are lethal if unrepaired
- Mismatch, base excision, and nucleotide excision repair use the other strand as a template
- Double-strand break repair includes:
- Homologous recombination
- Sister chromatid repair
- Non-sister repair
- Repair with exogenous DNA
- Nonhomologous end joining
Mechanism of Double-Strand Break Repair
- Processing of breaks occurs via 5' to 3' resection
- Creates a single-strand overhang with a free 3' OH group
- A single-strand overhang with a 3' OH group invades and base pairs with DNA on the homologous strand, forming a D-loop
- Requires recombinase
- The second strand with overhang and 3' OH invades the homologous strand
- DNA synthesis extends the 3' OH, using the homologous strand as a template
RecBCD Complex
- The RecBCD complex (helicase/nuclease) binds to double-stranded DNA ends and moves inward
- RecD helicase unwinds 5' to 3'
- RecB helicase unwinds 3' to 5'
- Nuclease degrades the unwound DNA
- RecC recognizes chi sites (5'-GCTGGTGG-3')
- Causes decreased degradation of the 3' end and increased degradation of the 5' end
- Produces single-stranded DNA with a 3' OH group
RecA Loading and Function
- RecA is loaded onto 3' extensions
- Nucleation is slow because single-stranded DNA is initially bound by SSB
- RecB recruits the first RecA
- Additional RecA rapidly binds cooperatively
- RecA coats the single-stranded DNA to form a RecA filament
- Enables the homology searches needed to repair double-stranded DNA breaks
- DNA-dependent ATPase
- Promotes strand invasion
- Helps single-stranded DNA find a complementary partner
- 3'-OH primer and single-stranded template allow DNA polymerase I to act
Synthesis-Dependent Strand Annealing (SDSA)
- Requires DNA polymerase and DNA ligase for complete replication and ligation
- Helicase melts complementary regions, leading to strand dissociation and annealing
Holliday junctions
- Holliday junctions
- Four-branched crossover junctions
- Branch points can slide back and forth at no energetic cost, known as branch migration
- Must be cut apart
- X and Y type cuts are equivalent in 3D
Resolution of Holliday Intermediates
- Option 1: Cut both junctions the same way to produce non-crossover "patch" products
- Option 2: Cut junctions differently to produce crossovers
- Enzymes like the RuvAB complex stimulate branch migration and resolution of Holliday junctions:
- RuvAB complex (repairs UV damage in E. coli)
- RuvA recognizes the Holliday junction and forms a complex with two RuvB hexamers
- RuvB is an ATPase that uses ATP hydrolysis to drive branch migration
- RuvC nuclease resolves Holliday junctions in E. coli
- RuvC is recruited to the RuvAB-Holliday junction and cleaves the DNA
- DNA ligase seals nicks
Enzymes/Proteins in Bacterial Recombinational DNA Repair
- RecA: Bacterial recombinase; filament on DNA
- RecB: Part of RecBCD heterotrimer; 3'→5' helicase, forms 3' strand extensions, also 5'→3' and 3'→5' exonuclease
- RecC: Part of RecBCD heterotrimer; binds chi, forms 3' strand extensions
- RecD: Part of RecBCD heterotrimer; 5'→3' helicase, forms 3' strand extensions
- RecF: Part of RecFOR mediator complex
- RecO: Part of RecFOR mediator complex; binds DNA, loads RecA onto ssDNA gap
- RecR: Part of RecFOR mediator complex
- RuvA: Tetramer; binds Holliday junctions, functions with RuvB
- RuvB: Hexamer; DNA translocase, promotes branch migration
- RuvC: Dimer; Holliday junction resolvase
- RecG: Monomer; helicase, promotes fork regression
- SSB: Tetramer; binds single-stranded DNA
- Pol I: Monomer; fills in gaps
- DNA ligase: Monomer; seals nicks
The SOS Response
- E. coli DNA repair is induced by DNA-bound RecA
- LexA constantly cycles on and off its binding site; abundant LexA ensures SOS genes are repressed
- RecA binds single-stranded (damaged) DNA
- DNA-bound RecA induces free LexA to cleave itself
- Lack of functional LexA allows SOS genes to be expressed
Meiosis and Recombination
- Double strand break repair is a key aspect of recombination during meiosis 1
- Creates genetic diversity and allows separation of favorable and unfavorable alleles
- Precisely aligned crossing over occurs during meiosis
Homologous Recombination in Meiosis
- Begins with a double strand break (DSB)
- Spo11 makes a double strand cut
- Spo11-DNA is removed by Mre11-Rad50-Xrs2
- Sae2 & Sgs1 remove more DNA from the 5'-end
- RecA class proteins are loaded onto 3'-end extensions
- Invading strand pairs with its complement, followed by DNA synthesis and ligation, forming a double Holliday intermediate
- Resolvases cleave the double Holliday intermediate
Recombination Outcomes
- Non-crossover events result in gene conversion
- Crossover events produce new combinations of genes
Physical vs. Map Distance
- Physical distance (in base pairs) can differ from map distance (in cM)
- Order of markers is the same on genetic and physical maps
- Frequency of recombination within a given DNA length can vary 100-fold
- Humans have approximately 30,000 hotspots spaced every 50-100 kb, correlated with nucleosome density, histone variants, euchromatin/heterochromatin, and chromosomal loops
Somatic (Mitotic) Recombination
- Non-sister (homologous) chromatids do not pair during mitosis, so do not undergo crossover and recombination
- Recombination can occur between non-sister chromatids at low frequency as a consequence of DNA repair mechanisms
- Allows an alternate way for double strand breaks to accurately align
- Can be problematic if a cell requires proper regulation of a gene
- Normal mitosis of a cell with one inactive copy of the Retinoblastoma gene (tumor suppressor) results in heterozygous daughter cells, where one wild type copy is sufficient
- Mitosis with crossover (somatic recombination) occurs more often in cancer cells → progressive loss of growth control
Gene Conversions
- Consequences of branch migration depend on the source of homologous DNA
- Sister chromatids: Should be identical except for mutations during replication
- Non-sister chromatids: Allelic differences can lead to mismatches in heteroduplex; can result in repair or gene conversion
- For homologous genes, distorts allelic frequency in progeny from expected Mendelian ratios
- In particular, duplicated genes provide the fuel for homologous recombination events that can cause inherited disease and cancer."
Replication Fork Issues
- When a replication fork arrives before damaged DNA and repair occurs
- Translesion synthesis
- Fork stalls
- Repair initiated
- Translesion DNA synthesis allows replication to continue
- TLS
- Lesion still needs to be fixed
- Replicative DNA polymerase can hop some minor types of damage
- Gap needs to be repaired later
- More frequent on lagging strand
- If DNA polymerase encounters a single strand region
- Fork collapses
- Can recover via a variation on DS break repair
- Stalling leads to Fork regression when DNA polymerase encounters a lesion
- Templates zipper back past lesion
- Daughter strands come together
- Occurs via MMR, NER, BER
- Daughter strands come together
- Templates zipper back past lesion
- After fork regression
- Lesion repair or Replication
CRISPR-Cas9
- It can be used to introduce double-strand cuts at precise locations in any gene in any organism, including humans.
- Plasmids containing the Cas9 gene are commercially available
- Cas9 plasmid (or Cas9 protein) can be introduced into cells along with the DNA targeting sequence
- Done by transformation or micro-injection into fertilized egg
- Via the addition of homologous donor DNA, the Cas9 double-strand cut can be repaired via homologous recombination
Applications of CRISPR-Cas9
- Used to replace one allele with another via the addition of homologous donor DNA
- Can easily be done with leaving no "fingerprints":
- Gene editing can happen in transiently
- Mutations are exact matches to naturally occurring DNA damage
- Difficult, if not impossible, to detect or block by regulation
- The technology is cheap and readily accessible
- ~ 50,000 people use it
Concerns with CRISPR
- MIT Technical review
- Chinese scientists created CRISPR babies in 2018 via inactivation of CCR5
- Live births of genetically modified humans created a storm of criticism • Concern over use of technology that can permanently change the human gene pool without adequate regulation and oversight.
- Chinese government has increased oversight
- Chinese scientists created CRISPR babies in 2018 via inactivation of CCR5
- Issues such as off-site changes are better understood/managed
- Can be difficult to treat in other ways
- Concern about impact on the slow and cautious work by others around the world
Nonhomologous End Joining
- Used as a last resort
- Enzymes involved in Nonhomologous End Joining:
- Ku70: Binds to DNA ends
- Ku80: Binds to DNA ends
- DNA-PKcs: Protein kinase catalytic subunit
- Artemis: Nuclease
- Pol μ: Fills in gaps
- Polλ: Fills in gaps
- XRCC4: Seals nicks
- XLF: Seals nicks
- DNA ligase IV: Seals nicks
- Is usually mutagenic, but useful for CRISPR/Cas gene inactivation
- Alternate nonhomologous end joining mechanism
Adaptive Evolution
- Does stress increase the rate of mutation and thus help cells develop resistance?
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